J. Embryol. exp. Morph. Vol. 18, 1, pp. 43-51, August 1967
With 1 plate
Printed in Great Britain
43
The experimental induction
of whisker growth in the hooded rat by
implantation of dermal papillae
By R. F. OLIVER 1
From the M.R.C. Unit for Research on the Experimental Pathology
of the Skin, University of Birmingham
Dermal papillae are regenerated and whiskers are produced after the experimental removal of up to the lower third of the vibrissa follicle, but if more of the
follicle is removed neither event occurs (Oliver, 1966 a, b). Similarly, when
lengths of the lower third of the vibrissa follicle wall are transplanted into ear
skin, whiskers are again produced, but not if lengths of wall are taken from
within the upper two-thirds of the follicle (Oliver, 1967). From these experiments it was clear that the outer root sheath and the adherent mesenchymal
layer, from which the new papillae are apparently derived, are the essential
tissues in the regeneration process.
The failure of papilla regeneration in the upper two-thirds of the follicle could
be explained in several ways. It is possible that the outer root sheath at this level
is incapable of supporting whisker growth or of stimulating papilla formation.
Alternatively, the cells of the mesenchymal layer at this level, unlike those in the
lower third of the follicle, may be incompetent to form a dermal papilla.
In an attempt to analyse these factors, vibrissa dermal papillae were implanted
into the bases of whisker follicles from which more than the lower third of the
root had been removed.
MATERIALS AND METHODS
Operation
All operations were performed on the vibrissa follicles on the upper lip of
animals 2^—3 months old from an inbred strain of hooded rat. Anaesthesia was
induced by the intra-peritoneal injection of a 10 x diluted solution of Nembutal
(Abbott), 0-65 ml/100 g body weight. The whisker roots were exposed for
operation by reflecting the whisker pad as previously described (Oliver, 1966 a).
1
Author's address: Medical Research Council, Unit for Research on the Experimental
Pathology of the Skin, Medical School, University, Birmingham 15, U.K.
44
R. F. OLIVER
The follicles selected for operation were identified and a record was made of the
length of the whiskers present in the follicles. Lengths of follicle were removed
with a transverse cut so as to leave half or less of the upper region of the
follicle in situ. The cut whisker shafts were then plucked from these follicle
remnants.
The removed lengths of follicle were placed in Hank's solution and the
bulbar ends isolated, once again, with a transverse cut. The dermal papilla was
dissected from each of these' end bulbs' using a method similar to that described
by Cohen (1961). Two opposing cuts were made in the length of the capsule to
the base of the bulb with a fine pair of iridectomy scissors and the two resulting
flaps of capsule reflected. Gentle pressure was exerted on the sides of the remaining part of the bulb to effect the separation of either the whole of the
ectodermal component from the dermal papilla or, more usually, just the matrix
leaving the dermal papilla within a tube of outer root sheath and the adherent
mesenchymal layer. This tube was reflected in an identical manner to the capsule. The dermal papilla was then very carefully 'cleaned', by scraping quite
firmly with a fine pair of watchmaker's forceps, under x 25 binocular vision, to
ensure that no matrix cells were left adherent to the surface of the papilla. The
dermal papilla was isolated from the rest of the bulb with a transverse cut across
its base which in most instances precluded the removal of the basal stalk since it
was not always possible to determine with certainty that all traces of the matrix
had been dislodged from the stalk. The dermal papilla was then placed on to the
cut surface of the follicle remnant and pushed gently into the mouth of the circular
tube provided by plucking the growing whisker. It was found impossible to
orientate the papillae in any particular way with certainty. In later operations
the keratinized inner lining of the follicle was broken up and removed as completely as possible with a fine pair of forceps before implanting the dermal
papilla. After implantation of the dermal papillae the lip flap was stitched back
in position. At no time after operation did the animals have difficulty in feeding
or drinking.
Experimental
Twenty-seven lengths of follicle were removed in a total of nine rats (three
follicles in each rat). Dermal papillae were successfully dissected from nineteen
of these lengths of follicle and implanted singly as autografts into nineteen of the
follicle remnants leaving eight as controls. Three rats received single implants,
two rats received two implants each and four animals received three implants
each.
The approximate stages of the growth cycle of all the follicles operated on
were determined by the lengths of the whiskers present in the follicles at operation and confirmed by the size and shape of the dermal papillae in those follicles
from which they were removed. Thus dermal papillae from known follicles and
at a known stage in the growth cycle were implanted into known follicle remnants also at a known stage in the growth cycle.
Induction of whisker growth
45
All the follicles, including those without papilla implants, were kept under
occasional observation for at least 110 days after operation and where whiskers
were produced their lengths were recorded. Four of the rats were then killed
112-141 days after operation and the operated follicles excised and prepared for
histological examination.
Identical operations were also performed on a further four rats, implanting
two or three dermal papillae in each animal, and these were killed at 1, 7,14 and
21 days after operation for information on the histological changes which occur
at early stages after implantation.
To determine whether, in fact, any matrix cells were left on the surface of the
papillae prior to implantation several dermal papillae which had been removed
routinely were prepared for histological examination.
Histology
Dermal papillae were fixed in Zenker's fixative and 5 fi serial sections were
stained with Ehrlich's haematoxylin and eosin. Operated follicles were fixed
in formol saline and serial sections 8/t in thickness were stained in a combination
of Weigert's haematoxylin, alcian blue and Curtis's Ponceau S.
RESULTS
None of the eight control follicles, those without papilla implants, produced
whiskers after operation. Of the nineteen follicles into which dermal papillae
were implanted, fourteen produced generations of whiskers which first appeared
above skin level between 18 and 25 days after operation.
The lengths of whiskers produced were extremely variable ranging from 14 to
73 % of the expected length for the various follicle positions. Although the
majority of implants were from late anagen donor follicles into late anagen
follicle remnants, the number of transplants made at other stages in the growth
cycle of either donor or recipient follicles were insufficient to permit any deduction as to the effect of this factor on subsequent whisker growth or whisker
length.
None of the papillae prepared for histological examination showed any
evidence of contamination with ectodermal matrix cells (Plate 1, fig. A.)
Nine follicles which had received grafts were examined histologically at early
intervals after operation. All nine of the implanted papillae had persisted and
were identified at histological examination. The exact level of removal of the
root ends was clearly indicated in all of the follicles by the cut edge of the
capsule and the abrupt termination of the thick glassy membrane.
In both follicles examined after 24 h the implanted papillae were present as a
discrete cell mass at the base of the follicle and isolated from the outer root
sheath by the keratinized inner root sheath (Plate 1,fig.B). The cytoplasm of the
papillae was stained blue-green indicating the presence of acid mucopoly-
46
R. F. OLIVER
saccharides, although the central region of one of the papillae, in which the
nuclei were more densely arranged, was less intensely stained.
By 7 days in the three follicles studied no traces of the inner root sheath were
present and the lower region of the outer root sheath had become a solid column
of cells which extended to just below the termination of the glassy membrane.
Dermal papillae were present as a distinct indentation at the most proximal end
of the outer root sheath in two of the follicles (Plate 1, fig. C), presenting a
slightly advanced stage in bulb development over the third follicle in which the
papilla was present as a cell mass at the flattened proximal surface of the outer
root sheath. Although in all three follicles the basal region of the outer root
sheath was markedly basophilic and contained cells in division, none of the
follicles had reached the stage of development of the hair cone. The nuclei of the
papillae were heavily stained and were more densely arranged than one day
after implantation showing a loss in overall papilla volume; none of the papillae
had taken up the alcian blue stain. More lightly stained nuclei, however, were
present in the most distal region of the papillae immediately adjacent to the
outer root sheath. The papillae, in which erythrocytes were present, were
encapsulated by a downgrowth from the cut edge of the mesenchymal layer.
PLATE 1
Fig. A. Vertical section of a dermal papilla dissected from the base of an anagen whisker
follicle. No epidermal matrix cells are attached to the surface of the papilla. Ehrlich's haematoxylin and eosin. x 230.
Figs. B-F. Longitudinal sections of the proximal end of whisker follicles at various stages
after implantation of dermal papillae into the bases of whisker follicle remnants after
removal of half or more of the lower region of the follicle. The level of removal is indicated
by the cut edge of the capsule.
Fig. B. Twenty-four hours after implantation of a dermal papilla (arrow). The papilla is
isolated from the outer root sheath by the keratinized inner root sheath. Weigert's haematoxylin, alcian blue and Curtis's Ponceau S. x 70.
Fig. C. Seven days after implantation of a dermal papilla. The inner root sheath has been
ejected distally and the lower region of the outer root sheath has become a solid column of
cells. The most proximal region of the outer root sheath is present as a shallow inverted
cup over the dermal papilla which has not taken up the alcian blue stain. Weigert's haematoxylin, alcian blue, Curtis's Ponceau S. x 55.
Fig. D. Fourteen days after implantation of a dermal papilla. The developing bulb projects
below the cut edge of the capsule and a whisker is being produced. The dermal papilla has
taken up the alcian blue stain. The abrupt termination of the thick glassy membrane indicates
the level of the original cut across the follicle wall. Weigert's haematoxylin, alcian blue and
Curtis's Ponceau S. x 60.
Fig. E. Twenty-one days after implantation of a dermal papilla. The follicle extends well
below the cut edge of the capsule. Weigert's haematoxylin, alcian blue and Curtis's Ponceau S.
x60.
Fig. F. One hundred and twenty-six days after implantation of a dermal papilla. A dermal
papilla is not present. Shown is a small sebaceous gland, whose duct opens into the follicle
cavity in adjacent sections, which has developed at the base of the follicle. Weigert's haematoxylin, alcian blue and Curtis's Ponceau S. x 200.
/. Embryo!, exp. Morph., Vol. 18, Part 1
R. F. OLIVER
PLATE 1
facing p. 46
Induction of whisker growth
47
By 14 days in both follicles examined the outer root sheath extended well
below the original cut across the follicle. A typical early anagen bulb was
present at the base of both follicles and whisker growth was in progress (Plate 1,
fig. D). The nuclei of the papillae were large and lightly stained and the cytoplasm had taken up the alcian blue stain. A well developed capillary system was
present in both papillae.
At 21 days one of the two follicles presented a very similar histological picture
to that described for the 14 days follicle and was producing a fine whisker shaft
(Plate 1, fig. E).
An extension of the follicle below the level of removal of the root end was not
present in the second follicle. The dermal papilla, which was stained blue-green
and contained capillaries, the matrix and the growing whisker shaft were not
arranged axially with respect to the length of the follicle segment. This follicle
was not producing a normal early anagen whisker fibre but a widely medullated,
thick, curved shaft without a cuticle which was fully keratinized at an abnormally
low level with respect to the bulb; peripheral to the apical region of the papilla
was a layer of living ectodermal cells 2-3 cells deep, and external to this was the
already keratinized shaft.
Four rats, in which three follicles in each rat had received papilla implants,
were killed for histological examination 112-141 days after operation. Of these
twelve follicles, four had not produced whiskers after operation and eight had
produced several generations of whiskers 7-22 mm in length, 14-52 % of the
club lengths normally produced by these follicles at the time of operation.
Histological examination confirmed that lengths of the root had been removed
in all of these follicles, as indicated by the cut across the capsule, from a level
within the inferior enlargement, just below the ring sinus, up to the level of the
ringwulst; that is, well within the region in which papillae are not regenerated
after simple removal of lengths of the follicle (a diagram of the structure of the
whisker follicle is presented in Oliver, \966b). There was no obvious correlation
between the level at which removal had occurred and the length of the
whiskers subsequently produced or even whether whiskers were produced or
not.
Seven of the eight follicles which had produced whiskers were in various
stages of anagen at the time of biopsy and one was in early catagen. In all cases
the follicle extended below the cut across the capsule. Seven of the eight follicles
had apparently normal looking bulbar regions, although one was producing a
whisker whose tip was spiralled within the follicle and had not penetrated the
skin surface. A spiralled whisker shaft was also found in association with another
follicle although at the time of biopsy the follicle was producing an apparently
normal whisker which extended above the skin surface. The spiralled whisker
had grown out of the side of the capsule into the surrounding connective tissue.
The eighth follicle, which had produced at least three generations of doubled
whiskers simultaneously, contained two bulbs from each of which was being
48
R. F. OLIVER
produced a single whisker. Both whiskers were contained within a common
outer root sheath higher in the follicle.
In three of the four follicle segments which had no history of whisker production after operation, the follicle cavity extended down to a level just above
the ringwulst and below this the outer root sheath was present as a solid column
of cells terminating at or just above the cut across the capsule. There was no
evidence of papilla cells or whisker production in any of the follicles. At the
base of one of the follicles, however, was a small sebaceous gland with a
sebaceous duct which opened into the bottom of the follicle cavity (Plate 1,
fig. F); a sebaceous gland was also present in the normal position at the neck
of the follicle.
The fourth follicle segment contained a remarkably distended thin walled
(1-2 cells thick) ectodermal sac which extended below the cut across the capsule.
Within the lumen of this sac were red-stained keratinized fragments and a
yellow-stained keratinized body which did not resemble, however, a whisker
shaft. In this follicle segment also there was no evidence of papilla cells or
whisker production.
DISCUSSION
Although it is generally assumed that the dermal papilla of the hair follicle
induces hair growth, conclusive evidence that the dermal papilla induces the
growth of epidermal appendages is only available for the feather follicle (Lillie
& Wang, 1941, 1944; Wang, 1943). They have shown that after removal of the
'whole papilla' (the dermal papilla and its ectodermal investment) from the base
of the feather follicle there is no further feather production and no regeneration
of a dermal papilla. However, if a dermal papilla is implanted into the basal
half of a follicle, from which the whole papilla has been removed, generations
of feathers are produced.
The present results clearly demonstrate that the dermal papilla is also the
inductive agent in the formation of the whisker bulb and in whisker growth.
Histological examination of the isolated dermal papillae has shown that the
technique used for their removal reliably ensures that no ectodermal matrix
cells from the donor follicle remain adherent to the papilla surface. None of the
eight follicle segments without papilla implants produced whiskers, confirming
the inability of the upper two-thirds of the follicle to regenerate papillae, and the
five which had received implants but which subsequently did not produce
whiskers showed no evidence of the presence of a papilla at biopsy—presumably
in these five follicles either the papillae did not remain in position after implantation or did not become successfully incorporated into the follicle and degenerated. Since whiskers were produced after the randomly orientated implantation
of papillae, often with the basal stalks absent, it seems possible that the implanted papilla interacts as a non-organized cell mass with the outer root sheath
to trigger the sequence of events leading to whisker growth. Among these events
Induction of whisker growth
49
are the reorganization of the papilla cell mass in its spatial relationship with the
ectodermal component of the follicle, the initial loss of staining for acid mucopolysaccharides and then the reappearance of acid mucopolysaccharides in the
papilla in the later stages of bulb development, and the extension of the follicle
below the level of root removal. The ejection of the inner root sheath as the outer
root sheath becomes a solid column of cells also occurs after removal of the
lower region of the vibrissa follicle prior to papilla regeneration (Oliver, 1966&),
but in this case there is little or no eventual growth of the follicle below the level
of root removal.
It is also clear that the non-regeneration of papillae and whiskers from the
upper two-thirds of the folhcle after simple removal of lengths of follicle arises
from the inability of the mesenchymal layer to form a papilla at this level and is
not due to the incompetence of the outer root sheath to become organized for
hair production. Although it seems most likely that it is the dermal element of
the follicle which initiates whisker growth it may be that it is the ectodermal
element which is the prime mover both in initiating the regeneration of papillae
and in the 'activation' of the resting papilla so that it induces renewed hair
growth in the normal hair growth cycle. In other words, the signal for the
mesenchymal layer to form a dermal papilla may still ultimately come from the
outer root sheath and in the upper two-thirds of the folhcle it is unable to give
this signal.
The design of the present experiment does not allow a conclusive explanation
for the variation in the lengths of whiskers produced. Lack of correlation
between the stages of the growth cycle of the donor and host follicles at the time
of operation and the lengths of whiskers produced does not provide any significant indications that this factor is important; however, a consistent length of
root was not removed from each follicle. Although there did not appear to be a
correlation between the level at which removal of the root occurred and the
length of the whiskers subsequently produced, the length of follicle into which
the papilla is implanted and the size (cell number) of the implanted papilla and
the degree of successful incorporation of the papilla into the follicle may well
be the most important factors in determining the length of whisker produced.
Wang (1943) has demonstrated that the feather dermal papilla cannot induce
feather growth from the distal or superficial half of the feather follicle nor when
implanted under extra-follicular epidermis. This contrasts with the vibrissa
papilla which can induce whisker growth from the distal half of the vibrissa
follicle. The question now arises as to whether the vibrissa papilla can also
induce hair follicle formation and hair growth when placed in contact with
extra-follicular epidermis, a possibility suggested by Billingham & Silvers (1963).
Experiments are now being performed to test this hypothesis.
JEEM
l8
50
R. F. OLIVER
SUMMARY
1. A method is described for isolating dermal papillae from the vibrissa
follicles on the upper lip of the hooded rat.
2. Dermal papillae have been implanted into the bases of the upper region of
vibrissa follicles which do not otherwise regenerate papillae and whiskers.
3. Fourteen out of nineteen of the follicle segments which received papilla
implants subsequently produced regenerations of whiskers. None of the eight
control follicle segments (those without papilla implants) produced whiskers.
4. The vibrissa dermal papilla was thus conclusively shown to be the inductive
agent in bulb formation and whisker growth.
5. The non-regeneration of papillae and whiskers from the upper two-thirds
of the follicle therefore arises from the inability of the mesenchymal layer to
form a papilla and is not due to the inability of the outer root sheath at that
level to become organized for whisker growth.
6. The dermal papillae, which were randomly orientated at implantation, lose
their initial organization by 7 days after implantation and then become incorporated in the bulb which develops at the base of the outer root sheath. The
follicle then grows down below the level of the root removal.
7. A conclusive explanation could not be offered for the variation in length
of the whiskers produced after implantation of dermal papillae.
RESUME
Induction experimentale de la croissance des vibrisses chez le
rat mantele par implantation de papilles dermiques
1. On decrit une methode d'isolement des papilles dermiques a partir des
follicules des vibrisses, sur la levre superieure du rat mantele.
2. On a implante des papilles dermiques dans la base de la region superieur
de follicules de vibrisses qui ne regenerent pas, d'autre part, de papilles ni de
vibrisses.
3. Sur 19 segments folliculaires ayant recu des implantations de papilles, 14
ont par la suite regenere des vibrisses. Aucun des 8 segments folliculaires
temoins (ceux qui n'avaient pas de papilles implantees) n'a produit de vibrisses.
4. On a done montre de maniere concluante que la papille dermique de la
vibrisse est l'agent inducteur de la formation du bulbe et de la croissance de la
vibrisse.
5. L'absence de regeneration de papilles et de vibrisses a partir des deuxtiers superieurs du follicule provient done de Pinaptitude de la couche mesenchymateuse a former une papille et n'est pas due a l'inaptitude de la gaine
externe de la racine, a ce niveau, a s'organiser pour la croissance d'une vibrisse.
6. Les papilles dermiques, qui ont ete orientees au hasard lors de Pimplantation, perdent leur organisation initiale sept jours apres cette derniere, puis
Induction of whisker growth
51
s'incorporent au bulbe qui se developpe a la base de la gaine externe de la racine.
Le follicule s'accroit alors au-dessous du niveau de l'exerese de la racine.
7. On n'a pu trouver d'explication concluante pour la variation de longueur
des vibrisses produites apres implantation de papilles dermiques.
I am most grateful to Dr C. N. D. Cruickshank, Director of this Unit, for his helpful
advice and criticism.
REFERENCES
R. E. & SILVERS, W. K. (1963). The origin and conservation of epidermal
specificities. New Engl. J. Med. 268, 477-80, 539^5.
COHEN, J. (1961). The transplantation of individual rat and guinea-pig whisker papillae.
J. Embryol exp. Morph. 9, 117-27.
LILLIE, F. R. & WANG, H. (1941). Physiology and development of the feather. V. Experimental morphogenesis. Physiol. Zool. 14, 103-33.
LILLIE, F. R. & WANG, H. (1944). Physiology and development of the feather. VII. An
experimental study of induction. Physiol. Zool. 17, 1-31.
OLIVER, R. F. (1966a). Whisker growth after removal of the dermal papilla and lengths of
follicle in the Hooded rat. /. Embryol. exp. Morph. 15, 331-47.
OLIVER, R. F. (19666). Histological studies of whisker regeneration in the Hooded rat.
/. Embryol. exp. Morph. 16, 231-44.
OLIVER, R. F. (1967). Ectopic regeneration of whiskers in the Hooded rat from implanted
lengths of vibrissa follicle wall. /. Embryol. exp. Morph. 17, 27-34.
WANG, H. (1943). Morphogenetic functions of the epidermal and dermal components of the
papilla in feather regeneration. Physiol. Zool. 16, 325-50.
BILLINGHAM,
{Manuscript received 12 January 1967)
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